The objectives of the proposed research are to investigate the mechanisms of (acyl) carnitine transport in natural and phospholipid-enriched inner mitochondrial membranes as well as in artificial membranes and reconstitued proteoliposomes, and to evaluate the role of this transport in control af fatty acid oxidation. The first specific aim of this proposal is to understand the role of membrane protein: lipid ratios in affecting diffusion versus carrier-mediated membrane transport of carnitine and fatty acylacarnitine. The kinetics of binding and free transmembrane diffusion will be followed in artificial lipid bilayers using the flourescent probes, pyrenebutyryl- or pyrenedecanoylcarnitine. The effects of phospholipid composition on free diffusion will be determined. Free and facilitated diffusion and carrier-mediated exchange activity will be studied in mitochondrial inner membranes in the native state and enriched by 30-700% with phospholipid (Schneider et al, Proc. Natl. Acad. Sci. USA 77, 1980; 442). The effects of the composition of phospholipid used for enrichment on carnitine and acylcarnitine transport will be determined in this system as well as in reconstituted proteoliposomes containing both carrier-mediated and facilitated difusion transport (Schulz and Racker, Biochem. Biphys. Res. Commun. 89, 1979:134). The second specific aim is to examine the role of the carnitine-acylcarnitine translocase for possible rate limitation of fatty acid oxidation using a specific inhibitor, pyrenebutyrylcarnitine and ligand-concentration plot analysis of ATP production and oxygen consumption. The effects of conditions in vivo which increase fatty acid oxidation (fasting, glucagon, diabetes) on translocase activity and matrix carnitine levels will be analyzed for changes in translocase activity and/or number of translocase sites. The possibility of changes in the diffusional component of carnitine uptake into the mitochondrial matrix will be investigated with mitochondrial inner membrane visicles from ketogenic rats when total mitochondrial carnitine increases. The role of carnitine and acylcarnitine transport in oxidation of fatty acids may be significant in the control of diabetic ketosis as well as in carnitine deficiency syndromes, e.g. cirrhosis, ischemia and specific myopathies.